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PREFORMULATION &

ANALYTICAL METHOD DEVELOPMENT &

Validation

Chapter 2: Preformulation Studies

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2.1. Preformulation and Standardization of Drugs

Preformulation is an exploratory activity that begins early in drug development.

Preformulation studies are designed to determine the compatibility of excipients with the

active substance for a biopharmaceutical, physicochemical, and analytical investigation

in support of promising experimental formulations. These studies are conducted to form

the basis for the rational of formulation design. Data from preformulation studies provide

the necessary groundwork for formulation attempts.

The selected drugs and excipients were standardized as per respective pharmacopoeial

specifications, or as per the manufacturers’ specifications. The Certificates of Analysis

provided by the suppliers in case of gift samples have been duly included in the

appropriate sections.

2.1.1. Mepivacaine HCl (MH)

Chemical Name: - (±)-1-Methyl-2′, 6′ pipecoloxylidide monohydrochloride

Molecular Formula: - C15H22N2O. HCl

Molecular Weight: - 282.81

Appearance: A white crystalline powder

Solubility: Freely soluble in water and methanol, very slightly soluble in methylene

chloride.

Melting point: 255 - 2620C with partial decomposition

2.1.2. Mepivacaine Base (MB)

Chemical Name: - N-(2, 6-dimethylphenyl)-1-methylpiperidine-2-carboxamide

Molecular Formula: - C15H22N2O

Molecular weight: - 246

Appearance: Odourless, white powder


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Solubility: Water solubility is 7000 mg/L. It is soluble in alcohol, methanol, chloroform,

acetone and phosphate buffer pH 6.8.

Melting point: 149-153°C

2.1.3. Lidocaine HCl

Chemical Name:- 2',6'-Acetoxylidide, 2-(diethylamino)-, hydrochloride

Molecular Formula: - C14H22N2O . HCl

Molecular weight: - 270.8

Appearance: White crystalline solid powder

Solubility: Very soluble in water and alcohol, soluble in chloroform, insoluble in ether

Melting point: 77°C

Dissociation constant: pKa is found to be 13.78

Indication: For production of local or regional anesthesia by infiltration techniques such

as percutaneous injection and intravenous regional anesthesia by peripheral nerve block

techniques such as brachial plexus and intercostal and by central neural techniques such

as lumbar and caudal epidural blocks.

Lidocaine HCl was used as a reference drug for comparative investigations on developed

formulations. Hence was procured, standardized and analytical method was developed.

2.2. Standardization of Drugs and Excipients

2.2.1. Monographic Evaluation of Mepivacaine HCl, Mepivacaine Base and

Lidocaine HCl

Mepivacaine base and its hydrochloride salt were procured from Hagzhou

Verychem Science and Technology Co. Ltd. China. Both the drug forms were

standardized as per USP monograph; purity and identity were checked with Certificates

of Analyses provided by supplier. Lidocaine HCl was procured as a gift sample from

Gufic Biosciences Limited, Mumbai. It was standardized as per monograph and purity

and identity were checked with Certificate of Analysis provided by supplier.

Mepivacaine HCl, Mepivacaine Base and Lidocaine HCl were tested for the following-


49

Appearance: Colour of drugs was observed visually.

Solubility: Solubility was checked in alcohol, methanol, chloroform, acetone and

phosphate buffers of different pH.

Identification tests: Infrared spectrum of drugs was investigated using FTIR Infrared

Spectrophotometer using potassium disk method. Spectrum was scanned over the

wave number range 4000-400 cm-1

.

Loss on drying: Drug (1gm) was weighed and dried in an oven at 100°C- 105°C to

constant weight for 4 hours. The weight was again recorded.

Melting point: This was determined using melting point testing apparatus.

Assay: Percent drug content was considered as mentioned in Certificate of Analysis

of drug obtained from the suppliers and confirmed by the analytical method described

in later section.

The results of the standardization tests are mentioned in Tables 2.1-2.5 & Figs 2.1-2.4.

Table 2.1: Monographic evaluation of Mepivacaine HCl

TESTS SPECIFICATIONS RESULTS

Appearance White or almost white powder Complied

Identification By IR Complied

Loss on drying (w/w) 1.0% max 0.1%

Melting point 260-2620C 262°C

Assay 98.5-101.0% 99.97%

Table 2.2: Monographic evaluation of Mepivacaine Base

TESTS SPECIFICATIONS RESULTS

Appearance White or almost white powder Complied

Identification By IR Complied

Loss on drying (w/w) 1.0% max 0.1%

Melting point 149–153°C 152°C

Assay 98.0% -102.0 % (On anhydrous basis) 99.87%


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Fig 2.1: Certificate of Analysis of Mepivacaine Base & HCl

Fig 2.2: IR spectrum of Mepivacaine HCl and Base

Table 2.3: I.R Frequencies for Mepivacaine HCl and Base

I.R. Frequencies obtained for

Mepivacaine HCl

Frequencies obtained for

Mepivacaine Base

Frequency [cm-1

] Assignment Frequency (cm-1

) Interpretation

2957.14 N-H stretch ~3000 N-H stretching

2488.40 C-H stretch ~2500 C-H stretching

1674.36 HN-C=O (amide) ~1650 HN-C=O(amide)

1537.40 C=C (aromatic) ~1550 C=C(aromatic)


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Table 2.4: Monographic evaluation of Lidocaine HCl

Tests Specifications Results

Appearance White or almost white crystalline powder Complied

Solubility Very soluble in water, freely soluble in chloroform and

in ethanol 95%. Practically insoluble in ether

Complied

Identification By IR, To match with working standard Complied

Assay 98.0% -102.0 % ( On anhydrous basis) 99.8%

pH 4-6 4.5

Clarity and

colour of

solution

A 5 %W/V solution in CO2 free water is clear Complied

Fig 2.3: IR spectrum of Lidocaine HCl

Table 2.5: Frequencies obtained for Lidocaine Base

Frequency (cm-1

) Interpretation

3248 cm-1

N-H stretch (sec amine)

1654 cm-1

C=O stretch

1505 cm-1

C=C (aromatic)


52

Fig 2.4: Certificate of Analysis of Lidocaine HCl

2.2.2. Standardization of Excipients

The choice of the excipients depends on several factors; namely, the drug used, the

process involved, the formulator and the cost of excipients. All the excipients used in the

formulation development were procured from authentic vendors and Certificates of

Analysis were obtained for the same. Some of the important tests were performed as per

monographs and Certificate of Analysis to confirm the quality of the excipients.

Majority of the excipients used for the formulation development work are as listed in

Table 2.6:


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Table 2.6: List of Excipients used in Formulation Development

Name of the Excipient Source Use

Ethanol 95% S. D Fine Chemicals, Mumbai. Co-solvent

Isopropyl alcohol S. D Fine Chemicals, Mumbai. Co-solvent, cosurfactant

Propylene glycol S. D Fine Chemicals, Mumbai. Co-solvent

Acetone S. D Fine Chemicals, Mumbai. Solvent

Triethanolamine S. D Fine Chemicals, Mumbai. Neutralizer

Hydroxypropyl

methylcellulose (HPMC)

Aqualon, Mumbai Gelling agent, polymer

Carbopol 980 NF Lubrizol, Mumbai Gelling agent, polymer

Carbopol 971P NF Lubrizol, Mumbai Gelling agent, polymer

Carbopol 974P NF Lubrizol, Mumbai Gelling agent, polymer

Transcutol P Gattefosse Ltd, Mumbai Co-surfactant

Labrasol Gattefosse Ltd, Mumbai Surfactant

Tween 80 S. D Fine Chemicals, Mumbai. Surfactant

Butylated hydroxylanisole S. D Fine Chemicals, Mumbai. Anti-oxidant

Butylated hydroxyltoluene S. D Fine Chemicals, Mumbai. Anti-oxidant

Sodium metabisulphite S. D Fine Chemicals, Mumbai. Anti-oxidant

Poloxamer F68 BASF, Mumbai Film forming polymer

Kollidon® 30 BASF, Mumbai Film forming polymer

Eudragit RL 100 Evonik Degussa, Mumbai Film forming polymer

Oleic acid S. D Fine Chemicals, Mumbai. Penetration enhancer &

oil

Menthol Sigma Aldrich Penetration enhancer

Eugenol Sigma Aldrich Penetration enhancer

Propyl paraben S. D Fine Chemicals, Mumbai. preservative

Methyl paraben S. D Fine Chemicals, Mumbai. preservative

1. Ethanol 95%

It is primarily used as a solvent and as penetration enhancer.

Table 2.7: Monographic evaluation of Ethanol 95%


Description

Clear, colorless and volatile liquid with

slight characteristic odor and burning taste

Complied

Non-volatile residue Not more than 1.0 mg 0.3 mg

Specific gravity 0.812-0.816 g/mL 0.814

g/mL


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2. Isopropyl alcohol (Propane-2-ol)

It is used in cosmetics and pharmaceutical formulations primarily as a solvent in

topical formulations.

Table 2.8: Monographic evaluation of isopropyl alcohol

Tests Observations Specifications and

remarks

Characteristics Clear colourless liquid Complied

Solubility Miscible with benzene, chloroform,

ethanol, ether, glycerine and water.

Soluble in acetone

Complied

Boiling point 81.2 oC Confirmed (82.4

oC)

Specific gravity 0.788 Confirmed (0.786)

3. Propylene glycol

It is widely used as water miscible co-solvent, and penetration enhancer in topical

formulations.

Table 2.9: Monographic evaluation of Propylene glycol


Description Clear, colorless, viscous, practically

odorless liquid with a sweet, slightly

acrid taste resembling that of glycerin.

Complied

Residue on ignition ≤3.5 mg 2.9 mg

Specific gravity 1.035–1.037 g/mL 1.035 g/mL

4. Hydroxypropyl methylcellulose (HPMC) USP

Hydroxypropyl methylcellulose (HPMC) is partially o-methylated and O-(2

hydroxypropylated) cellulose. HPMC is an odourless and tasteless, creamy-white

coloured fibrous or granular powder. It is available in various grades that vary in

viscosity and extent of substitution. It is used as a suspending and thickening agent in

topical formulations. It has found major application as an emulsifier, suspending agent,

and stabilizing agent in topical gels and ointments.


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Table 2.10: Standardization of HPMC K4M USP XXIV

Test Observation Specification and

Inference

Characteristics Odourless, tasteless, white granular

powder.

Complied

Solubility Soluble in cold water forming a viscous

solution. Insoluble in alcohol, ether,

chloroform.

Complied

Apparent viscosity

cPs (USP)

4000 Passes

Loss on drying (%) 2.5 Passes (NMT 5%)

Sieve analysis (%)

% Through#40

% Through#100

99.8

94

Min 99

Min 90

pH, in water (1%) 7 Passes (5-8)

5. Tween 80 (Polysorbate 80) USP

Tween 80 is a non ionic surfactant and emulsifier derived from polyethoxylated sorbitan

and oleic acid. It is viscous, water soluble yellow liquid. The hydrophilic groups in this

compound are polyethers also known as polyoxyethylene groups which are polymers of

ethylene oxide.

Table 2.11: Standardization of Tween 80

Test Observations Specifications and Inferences

Appearance Yellowish Liquid Complied

Solubility Water soluble Complied

pH 4.6 4.5 – 5.5, Complied

Residue on Ignition 0.2% ≤0.25%

6. Carbopol 980 NF

Carbopol® 980 polymer is high molecular weight crosslinked polymer of acrylic acid,

which confirms to USP/INF specifications. Carbopol® 980 NF polymer is a highly

efficient thickener and it is ideal for formulating clear aqueous and hydroalcoholic gels.


56

Fig 2.5: Certificate of Analysis of Carbopol 980 NF

7. Carbopol 971P NF

Carbopol® 971P NF polymer is a lightly crosslinked polymer with long rheology, which

will result in flow like honey in a semisolid formulation. Carbopol 971P NF polymer can

be used to formulate low viscosity lotions, gels and liquids with good clarity.


57

Fig 2.6: Certificate of Analysis of Carbopol 971P NF

8. Carbopol 974P NF

Carbopol® 974P NF polymer is a highly crosslinked polymer and produces highly

viscous gels. Carbopol 974P NF polymer can be used to formulate viscous gels,

emulsions and suspensions.

Fig 2.7: Certificate of Analysis of Carbopol 974P NF


58

9. Transcutol P (Diethylene Glycol monoethylether) USP:

Transcutol P is a colorless liquid. It is soluble in both water and oil. It is used as a

cosurfactant, solubilizer and penetration enhancer in oral, rectal, vaginal, topical and

nasal delivery systems. It is a high performance solubilizer/solvent for many poorly

soluble compounds. It can be incorporated into all types of emulsions, solutions and gels.

10. Labrasol (Caprylocaproyl polyoxyl-8 glycerides NF)

A non-ionic water dispersible surfactant composed of well-characterised polyethylene

glycol (PEG) esters, a small glyceride fraction and free PEG. An oil-in-water surfactant

used to solubilize active pharmaceutical ingredients and promote drug penetration and

permeation.

11. Lutrol ® F68 (Poloxamer 188)

Lutrol ® F68 (Poloxamer 188) is a difunctional block copolymer surfactant terminating

in primary hydroxyl groups. It is a nonionic surfactant that is 100% active and relatively

nontoxic.

Fig 2.8: Certificate of Analysis of Transcutol P & Labrasol


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Fig 2.9: Certificate of Analysis of Poloxamer 188 (Lutrol ® F68)

12. Oleic acid

Oleic acid is a fatty acid that occurs naturally in various animal and vegetable fats and

oils. In chemical terms, oleic acid is monounsaturated omega-9 fatty acid. Oleic acid is

used as an excipient in pharmaceuticals and as an emulsifying or solubilizing agent in

aerosol products. It is also used as penetration enhancer in topical formulations.

Table 2.12: Monographic evaluation of Oleic acid


Description Colorless to light yellow liquid Complied

Odor Peculiar Lard-Like odor Complied

Boiling Point 286.11°C (547°F) 285.5

Specific Gravity 0.895 0.889

13. Kollidon® 30 (Polyvinylpyrrolidone)

Polyvinylpyrrolidone (PVP), also commonly called Polyvidone or Povidone, is a water-

soluble polymer made from the monomer N-vinylpyrrolidone. When dry it is a light flaky

powder, which readily absorbs up to 40% of its weight in atmospheric water. In solution,

it has excellent wetting properties and readily forms films.


60

Fig 2.10: Certificate of Analysis of Kollidon® 30 (Polyvinylpyrrolidone)

14. Eudragit RL 100 (Ammonio Methacrylate Copolymer Type A)

Eudragit® RL is copolymer of ethyl acrylate, methyl methacrylate and a low content of a

methacrylic acid ester with quaternary ammonium groups (trimethylammonioethyl

methacrylate chloride). The ammonium groups are present as salts and make the

polymers permeable.


61

Table 2.13: Monographic evaluation of Eudragit RL 100

Tests

Specifications Results

Description Colourless, clear to cloudy granules with a faint

amine-like odour.

Complied

Solubility

Soluble in methanol, ethanol and isopropyl alcohol

Soluble in acetone, ethyl acetate and methylene

chloride to give clear to cloudy solutions.

Complied

Residue on

evaporation Not less than 97.0 % Passes

Loss on drying Max. 3.0 % 2.8

Apparent

viscosity cP Max. 15 13.9

15. Menthol

Menthol is an organic compound made synthetically or obtained from peppermint or

other mint oils. It is a waxy, crystalline substance, clear or white in color, which is solid

at room temperature and melts slightly above. Menthol has local anesthetic and counter

irritant qualities. It is also used as penetration enhancer.

Table 2.14: Monographic evaluation of Menthol


Description Solid crystalline, white/clear in color Complied

Odor Characteristic of menthol, camphorous Complied

Melting Point 42°C (107.6°F) 41.7

Specific Gravity 0.89 0.87

16. Eugenol

Eugenol is an allyl chain-substituted guaiacol (2-methoxyphenol). It is an active element

in clove. It is also used as penetration enhancer. Its properties make it a good local

antiseptic and analgesic. It also makes a good local anesthetic for temporary relief from

toothache pain

Table 2.15: Monographic evaluation of Eugenol


Description Clear or pale yellow oily liquid Complies

Odor Aromatic smell typical of cloves Complies

Solubility Slightly soluble in water and soluble in organic solvents Complies

Boiling Point 252° C 251.5


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2.3. Container Closure System

Based on literature search on packaging systems considered for dispensing

pharmaceutical gels, we used Aluminum collapsible tubes lacquered internally and

Lamitubes (Provided as Gift sample from Essel Propack, Mumbai).

Fig 2.11: Tubes for packaging formulated Gels

2.3.1. Lamitubes

Lamitubes are more resistant to air and moisture. Lamitubes – ABL: Aluminum Barrier

Laminates is used. The multiplayer tubes (lamitube) made from laminates with

aluminium foil barrier combine the excellent barrier advantages of traditional metal tubes

& the attractive visual and tactile feel of the plastic tubes. The lamitube body and the

shoulder provide excellent barrier properties against permeation of gases like oxygen.

The tube, a soft squeezable container which can be used for gels and creams, is made of

laminated material to protect the contents. The tube is hermetically sealed and almost

sterile as a result of exposure to high temperatures during the production process. The

tube has special coatings to prevent the material from reacting with the contents. The

most important feature of these tubes is the barrier properties which keep the contents

safe from the atmosphere. With their high stability, laminated tubes are suitable for

packaging a variety of products. ABL tubes, is a customized specialty laminates, having

an aluminum foil barrier, which provides superior light, air and moisture barrier along

with reduced flavor absorption. It has tamper evident closure with nozzle seal. The

material density offers a more durable tube and allows for additional dispensing of the

products contents. Pastes, ointments, cream and gels typically dentifrice, over-the-counter

and pharmaceutical products fare well in ABL tube packaging.

These lamitubes were used for packaging and dispensing of gels and were compared with

the Aluminum collapsible tubes available as alternative packaging option.


63

2.3.2. Aluminum Collapsible Tubes

Aluminum collapsible tube is the only safe and secure means of packaging for cream,

ointment, cosmetics and other products. Aluminium tubes can be well decorated

externally and are coated internally to provide lasting stability. Impermeable nature of

aluminium collapsible tubes protects the inside product from oxidation and its collapsible

nature does not allow air contamination once opened. Aluminium collapsible tubes have

close mouth feature thus ensuring effectiveness and integrity of the product. Its seamless

and jointless nature also imparts leak proof property. Aluminum collapsible tubes of

various capacities (7 g, 10 g and 20 g) were obtained as gift sample from D. J. Industries,

Mumbai and were used for packaging and dispensing of gels.

Fig 2.12: Certificate of Compliance of Aluminum collapsible tube


64

2.4. Results

The standardization of drugs and excipients is an integral part of any research work and

ensures quality of the research outcomes.

Mepivacaine HCl and base was standardized as per the specifications given in the

monograph in USP30 NF25, 2007. Tables 2.1 and 2.2 enlist the various tests,

observations and specifications. The COA obtained from the supplier is shown in Fig 2.1.

The drug passed all the tests for identity and was well within pharmacopoeial limits. The

FT-IR spectra’s are shown in Figure 2.2

Lidocaine HCl was standardized as per the specifications given in the monograph in

USP30 NF25, 2007. Table 2.4 enlists the various tests, observations and specifications.

The drug passed all the tests for identity and was well within pharmacopoeial limits. The

FT-IR spectrum is shown in Figure 2.3.

Solvents such as Ethanol 95%, isopropyl alcohol and propylene glycol were standardized

as per the specifications of IP’2007. Tables 2.7, 2.8 and 2.9 enlist the various tests,

observations and specifications.

Polymers such as HPMC K4M, Carbopol 980 NF, Carbopol 971P NF, Carbopol 974P

NF, Kollidon® 30 and Eudragit RL 100 were standardized as per the specifications.

Table 2.10, Figures 2.5, 2.6, 2.7, 2.10 and Table 2.13 enlist the various tests,

observations and specifications respectively. The polymer passed all the tests for identity

and was well within pharmacopoeial limits.

Poloxamer F68 was standardized as per the specifications of USP’30. Figure 2.9 shows

the various tests, observations and specifications. The excipient passed all the tests for

identity and was well within pharmacopoeial limits.

Penetration enhancers such as menthol and eugenol were standardized as per the

specifications of USP’30. Tables 2.14 and 2.15 enlist the various tests, observations and

specifications. They passed all the tests for identity and were well within pharmacopoeial

limits.

Oleic acid was standardized as per the specifications of USP’30. Table 2.12 enlists the

various tests, observations and specifications. The excipient passed all the tests for

identity and was well within pharmacopoeial limits.


65

Surfactants such as Tween 80, Transcutol P and Labrasol were standardized as per the

specifications of USP’30. Tables 2.11 and Figure 2.8 enlist the various tests,

observations and specifications. The surfactant passed all the tests for identity and was

well within pharmacopoeial limits.

Packaging and dispensing tubes, lamitube and aluminum collapsible tubes of various

capacities were procured from authentic suppliers. Figure 2.12 shows the Certificate of

Compliance of Aluminum collapsible tube and its cap obtained from the manufacturer.

Lamitube of capacity 20g was used as supplied from manufacturer.

The standardized drugs and excipients were used in different permutations to develop

various formulations such as gels, nanoemulsions and metered dose topical spray

formulations as described in the following chapters.


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2.5. Analytical Method Development and Validation

2.5.1. Introduction

Analytical method development and validation play an important role in the formulation

and manufacture of pharmaceuticals. For routine analysis of drug in respective

formulations, assessment of drug content in the developed formulations, to detect and

quantify the drug in the presence of other constituents of the formulation and

investigation of release of drug from the developed formulations, suitable analytical

method needs to be developed and validated. For this purpose following analytical

methods were developed.

2.5.2. Analytical Method Validation

Validation of analytical methodologies is widely accepted as pivotal before they are put

into routine use. A method must be tested for effectiveness and must be appropriate for

the particular analysis to be undertaken. Method validation is defined as the process of

proving, through scientific studies, that an analytical method is acceptable for its intended

use and it instills confidence that the method can generate test data of acceptable quality.

The International Conference on Harmonization has issued guidelines for analytical

method validation [ICH Q2 (R1)].

Linearity and range

Linearity defines the analytical response as a function of solute concentration and range.

It prescribes a region over which acceptable linearity, precision and accuracy are

achieved. Linearity is generally reported as the variance of the slope of the regression

line. Range is the interval between the upper and lower concentrations of solute that have

been demonstrated to be determined with precision, accuracy and linearity. The ICH

guidelines specify a minimum of five concentrations, along with certain minimum

specified ranges

Accuracy and bias

Accuracy is the measure of exactness of an analytical method, or the closeness of

agreement between the measured value and the value that is accepted either as a

conventional, true value or an accepted reference value. Bias assesses the influence of the

analyst on the performance of the method.


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Precision

Precision quantifies the variability of an analytical result as a function of operator,

method manipulations and day-to-day environment. It is also the measure of the degree of

repeatability of an analytical method under normal operation and is expressed as the

percent RSD for a statistically significant number of samples. Precision experiments give

a good indication of the performance of the method and should be repeated regularly.

Generally, any increase of the RSD above 2.0% should be investigated. According to

ICH, three types of precision can be defined and should all be assessed as described

below.

Repeatability: It refers to the results of the method operating over a short time

interval under the same conditions (inter-assay precision). It expresses the degree of

variation arising during replicate assays performed consecutively and non-

consecutively but on the same day. Repeatability should be determined from a

minimum of nine determinations covering the specified concentration range of the

procedure.

Intermediate precision (ruggedness): It refers to the results from laboratory

variations due to random events such as differences in experimental periods, analysts

and equipment.

Reproducibility: It is an indication of the ability of the method to be transferred from

one laboratory to another.

Specificity and selectivity

A method is specific if it produces a response for only one single solute. Since it is almost

impossible to develop a chromatographic assay for a drug in a matrix that will respond to

only the compound of interest, the term selectivity is more appropriate. Selectivity

describes the ability of an analytical method to differentiate various substances in the

sample and is applicable to methods in which two or more components are separated and

quantitated in a complex matrix. It is a measure of degree of interference from such

things as other active ingredients, excipients, impurities and degradation products,

ensuring that a peak response is due only to a single component, i.e., that no co-elution

exists.


68

Limit of Detection and Limit of Quantitation

The USP requires that the limit of detection (LOD) and the limit of quantitation (LOQ)

be determined for studies that involve the detection and quantitation of components at or

near trace levels. The LOD is defined as the lowest concentration of a solute in a sample

that can be detected, though not necessarily quantitated and the LOQ is defined as the

lowest concentration of a solute in a sample that can be determined with acceptable

precision and accuracy under the stated operational conditions of the method. LOD was

based on determination of standard deviation of the response and the slope of the

corresponding curve using the following equation-

LOD = 3 s/m

Where s, the noise of estimate, is the standard deviation of the absorbance of the sample

and m is the slope of the related calibration graphs.

LOQ was based on determination of standard deviation of the response and the slope of

the corresponding curve using the following equation-

LOQ = 10 s/m

Where s, the noise of estimate, is the standard deviation of the absorbance of the sample

and m is the slope of the related calibration graphs.

2.5.3. UV–visible spectrophotometric method for estimation of Mepivacaine HCl

and Mepivacaine base for drug assay in semisolid dosage form

The technique of ultraviolet-visible spectroscopy is one of the most frequently employed

in pharmaceutical analysis. It involves the measurement of the amount of ultraviolet-

visible (190 - 600 nm) radiation absorbed by a substance in solution. Presently, the use of

ultraviolet spectrophotometry as an analytical procedure has become less popular due to

the advent of more sophisticated techniques such as chromatographic separation

techniques resulting in rare literature been available for the determination of mepivacaine

in pharmaceutical formulations. Hence, an attempt was made to develop and validate an

ultraviolet absorption spectrophotometric method for the determination of mepivacaine in

topical formulations and compare the outcomes to those obtained from HPLC.

The analytical procedure employed for quantitation of mepivacaine HCl and mepivacaine

base by U.V. spectrophotometry was determined in phosphate buffer solution (PBS) pH

6.8 and methanol.


69

2.5.3.1. Method Development:

Mepivacaine HCl and base solutions (100 μg/ml) both in methanol and phosphate buffer

solution pH 6.8 were scanned in the range of 200-400 nm using JASCO Ultraviolet

Spectrophotometer. The absorption maximum was obtained at 263 nm for phosphate

buffer pH 6.8 as well as for methanol are as shown in the figure 2.14 and 2.16. Hence

calibration curve was obtained using both the solvents. The following spectrophotometric

assay method was developed for analysis of the drug in the topical formulations:

Accurately weighed 100mg of drug was dissolved in sufficient amount of phosphate

buffer solution (PBS) pH 6.8 (or methanol) in a 100ml volumetric flask and diluted to

volume with phosphate buffer pH 6.8 (or methanol) so as to obtain solution of

concentration 1000μg/ml. From this solution, aliquots of 1.5, 2, 2.5, 3, 3.5, 4 and 4.5 ml

were withdrawn in 10 ml volumetric flasks and diluted to volume with PBS pH 6.8

solutions (or methanol) so as to obtain standard solutions of concentrations 150, 200, 250,

300, 350, 400 and 450 μg/ml respectively. The absorbance of the standard solution was

determined on U.V. Spectrophotometer at 263 nm. In order to determine the repeatability

of the method, standardization of the drug was carried out in triplicates. A standard plot

of absorbance verses concentration of drug in µg/ml was obtained. The results are as

given in Table 2.16 and 2.17.

Table 2.16: Standard calibration data for MH and MB in PBS pH 6.8 at 263 nm

Concentration (µg/ml) Absorbance at 263 nm

Mepivacaine HCl Mepivacaine Base

150 0.2297± 0.03 0.2814± 0.03

200 0.3413± 0.03 0.3889± 0.02

250 0.4174± 0.02 0.4873± 0.03

300 0.4981± 0.02 0.5915± 0.03

350 0.5585± 0.05 0.657± 0.05

400 0.6305± 0.04 0.753± 0.04

450 0.6933± 0.03 0.8419± 0.04


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Fig 2.13: Standard Calibration curve of Mepivacaine HCl & Base in PBS pH 6.8

Fig 2.14: UV Scan of Mepivacaine HCl and Base in PBS pH 6.8

Table 2.17: Standard calibration data for MH and MB in Methanol at 263 nm



150 0.2254± 0.05 0.2601± 0.05

200 0.3241± 0.05 0.3381± 0.05

250 0.3982 ± 0.04 0.3986 ± 0.04

300 0.4694 ± 0.05 0.4774 ± 0.05

350 0.5399 ± 0.03 0.5491 ± 0.03

400 0.6115 ± 0.04 0.6205 ± 0.04

450 0.6992 ± 0.03 0.7039 ± 0.03


71

Fig 2.15: Standard Calibration curve of Mepivacaine HCl and Base in Methanol

Fig 2.16: UV Scan of Mepivacaine HCl and base in Methanol

2.5.3.2. Validation of developed UV Spectrophotometric method of Mepivacaine

The spectrophotometric method selected for the analysis of mepivacaine HCl and

mepivacaine base was validated for linearity, precision, accuracy, limit of detection, limit

of quantification and specificity.

1) Linearity:

Linearity was determined by making three calibration curves. For preparation of each

calibration curve, three calibration standard solutions were prepared at concentrations

ranging from 150- 450 μg/ml of mepivacaine. Absorbance of each standard solution was

measured at 263nm. The absorbance was plotted against the corresponding

concentrations to obtain the calibration graph and was treated by linear regression

analysis. The calibration curves were found to be linear in the aforementioned

concentrations. The correlation coefficient (r²) of determination was 0.999 in phosphate

buffer. The results of test for linearity were evaluated using the regression line equation


72

obtained by the method of least squares. The ideal r² value should be more than or equal

to 0.998.

2) Accuracy:

Accuracy of the method was studied by recovery experiments. Accuracy is the percent of

analyte recovered by assay from a known added amount. Results are given in Tables 2.18

and 2.19.

Table 2.18: Intra-day and Inter-day accuracy of UV method developed for MH

Intra day Inter day

Added(μg/ml) Measured

(μg/ml) % recovery Added(μg/ml)

Measured

(μg/ml) % recovery

10 9.97 99.7 10 9.86 98.6

20 20.01 100.05 20 19.58 97.9

30 29.78 99.26 30 29.61 98.69

Table 2.19: Intraday and Inter-day accuracy of UV method developed for MB

Intra day Inter day


(μg/ml) % recovery Added(μg/ml)

Measured

(μg/ml) % recovery

10 9.95 99.5 10 9.83 98.3

20 19.96 99.8 20 19.68 98.4

30 29.68 98.92 30 29.58 98.59

3) Precision:

It was determined by repeatability (intraday) and intermediate precision (inter-day) and

reported as % RSD for a statistically significant number of replicate measurements. The

intermediate precision was studied by comparing the assays on three different days and

the results were documented as the standard deviation and % RSD. Precision of assay

(intraday precision i.e. repeatability) was evaluated by carrying out three independent

assays of test samples of mepivacaine HCl and base. The intermediate precision (inter-

day precision) of the method was also evaluated using two different analysts, systems and

different days in the same laboratory. The results are given in Tables 2.20 and 2.21.

Table 2.20: Intraday and Inter-day precision of UV method developed for MH

Intra day Inter day


(μg/ml) % RSD Added(μg/ml)

Measured

(μg/ml) % RSD

10 9.47 0.74 10 9.66 0.76

20 20.01 0.77 20 19.58 0.62

30 29.78 0.64 30 29.61 0.60


73

Table 2.21: Intraday and Inter-day precision of UV method developed for MB

Intra day Inter day

Added (μg/ml) Measured

(μg/ml) % RSD Added (μg/ml)

Measured

(μg/ml) % RSD

10 9.59 0.74 10 9.58 0.75

20 19.98 0.76 20 19.64 0.62

30 29.63 0.63 30 29.55 0.59

4) Limit of Detection

This is the minimum concentration of mepivacaine HCl and mepivacaine base that can be

detected but could not be quantified. The concentration at which smallest peak in the

spectrum was observed, was noted. The corresponding peak concentration of

mepivacaine HCl and mepivacaine base determined from the calibration curve was found

to be 20μg/ml.

5) Limit of Quantification

It is the minimum concentration of mepivacaine that can be quantified. The smallest peak

of mepivacaine HCl and mepivacaine base that could be detected and quantified in the

spectrum was determined. The LOQ was found to be 50μg/ml.

6) Specificity:

The specificity indicates that there was no interference observed during the test.

Table 2.22: Validation data of developed UV method for Mepivacaine HCl

Analytical Parameters Results

LOD(μg/ml) 20

LOQ(μg/ml) 60

Linearity

Range(μg/ml) 150-450

Slope ±% RSD 0.001±0.005

System precision

Amount taken(μg/ml) 10.00

Amount detected (μg/ml) ±% RSD 9.978±0.7


74

Table 2.23: Validation data of developed UV method for Mepivacaine Base


LOD (μg/ml) 20

LOQ (μg/ml) 60

Linearity

Range (μg/ml) 150-450

Slope ±% RSD 0.001±0.005

System precision

Amount taken (μg/ml) 10.00

Amount detected (μg/ml) ±% RSD 9.965±0.7

2.5.4. UV–visible spectrophotometric method for estimation of Lidocaine HCl

The following spectrophotometric assay method was developed for analysis of the

Lidocaine HCl: Accurately weighed 100mg of drug was dissolved in sufficient amount of

phosphate buffer pH 6.8 in a 100ml volumetric flask and diluted to volume with

phosphate buffer pH 6.8 so as to obtain solution of concentration 1000μg/ml. From this

solution, aliquots of 1.5, 2, 2.5, 3, 3.5, 4 and 4.5 ml were withdrawn in 10 ml volumetric

flasks and diluted to volume with phosphate buffer pH 6.8 solutions so as to obtain

standard solutions of concentrations 150, 200, 250, 300, 350, 400 and 450μg/ml

respectively. The absorbance of the standard solution was determined on U.V.

Spectrophotometer at 264 nm. In order to determine the repeatability of the method,

standardization of the drug was carried out in triplicates. A standard plot of absorbance

verses concentration of drug in µg/ml was obtained. The results are as tabulated.

Fig 2.17: UV Scan of Lidocaine

HCl in PBS pH 6.8 Fig 2.18: Standard Calibration

curve of Lidocaine HCl


75

Table 2.24: Standard calibration curve for Lidocaine in PBS pH 6.8 at 264 nm


150 0.2677

200 0.3589

250 0.4468

300 0.5362

350 0.6111

400 0.7050

450 0.7832

2.5.5. High Performance Liquid Chromatography (HPLC) Method for Estimation

of Mepivacaine HCl and Mepivacaine Base

HPLC provides rapid high resolution of compounds resulting in an efficient method of

analysis which can be completed over relatively short periods of time. In addition to this,

coupling HPLC to suitable detection methods provides increased sensitivity and

selectivity to facilitate accurate, precise and reproducible analysis of pharmaceutical

products. HPLC technique is based on the separation of components of a mixture by

virtue of differences in the equilibrium distribution of the components between

stationary and the mobile phase. The compound partitions between the different phases

based on their physicochemical properties and affinity for either of the phases. This

results in compounds eluting at different times and the consequent separation of the

drugs and components. The design of a successful HPLC separation method depends on

matching the right mobile phase to a given column and sample. Solvents used should be

readily available, compatible with the detector, safe to use, pure and relatively

unreactive. The solvent should be able to dissolve the sample.

For determination of concentration of Mepivacaine (Base & HCl) in formulation and in

skin layers, HPLC was performed using mobile phases as reported in the literature [Gill

R; 1984, GuCvello P. L; 1993, Einosuke T; 2006, Arie A.V; 1996].


76

Table 2.25: Mobile phases tried for HPLC analysis of Mepivacaine

Sr

No

Wavelength of

detection

Flow rate

(ml/min) Mobile Phase

1 210 nm 1.1

6.8:93.2 (v/v) isopropanol-sodium hydrogen

phosphate buffer solution with the pH adjusted to

6.8 using phosphoric acid.

2 210 nm 1.0 acetonitrile–methanol–30mM NaH2PO4 (pH 5.6)

(1:1:3, v/v/v).

3 210 nm 1.0 acetonitrile–methanol–30mM NaH2PO4 (pH 5.6)

(1:1:3: 0.05, v/v/v).

4 205 nm 1.0 0.01 M sodium dihydrogenphosphate (pH 2.1)-

acetonitrile (80:20, v/v).

5 210 nm 1.0 Methanol: water: 1% v/v solution of phosphoric

acid: hexylamine (30:70:100:1.4, v/v)

Acetonitrile and methanol, the two commonly used solvents, have ultraviolet cut-off

wavelength values of 190 and 205 nm respectively when highly purified. However the

peak obtained for mepivacaine was not distinct and poor resolution was obtained. Hence

mobile phase was developed for mepivacaine HPLC analysis. Sharp peaks that were well

resolved from the solvent front were observed with mobile phases comprising acetonitrile

and phosphate buffer solution pH 6.8 value.

2.5.5.1. Analytical Method Development for Assay of Mepivacaine

The aim of the investigation was to develop a method for the assay of drugs, Mepivacaine

HCl and Mepivacine base from the developed topical formulations.

Chromatographic conditions

An overview chromatographic condition of the HPLC method for Mepivacaine

quantification is as follows:


77

Column C18 Gold column (46mm x 250 mm) 2.5 µm

Mobile Phase Acetonitrile: Phosphate Buffer Solution pH 6.8 (6:4)

Flow rate 1 ml/min

Run time 15 min

Injection volume 20 µl

Mepivacaine quantification

wavelength 210 nm

Calibration curve for Mepivacaine HCl & Base: - Mepivacaine HCl or base (10mg)

was taken in 100 ml volumetric flask and diluted to volume with phosphate buffer

solution pH 6.8 and used as stock solution for preparation of calibration curves. The

reference solution was diluted with PBS to obtain concentration ranges of Mepivacaine

HCl/base (0.5-5 µg/ml). The calibration curves were plotted as peak area vs.

concentration (Fig. 2.6.9 & 2.6.10).

Table 2.26. Standard calibration data of MB in PBS pH 6.8

Concentration

(µg/ml)

Area


0 0 0

0.5 30.57 31.26

1 65.32 68.54

2 121.39 125.78

3 187.34 191.32

4 241.28 250.11

5 299.12 302.76

Fig 2.19: HPLC Scan of MH in

PBS in pH 6.8

Fig 2.20: HPLC Scan of MB in PBS in

pH 6.8


78

2.5.5.2. Analytical Method Development and Validation for Quantification of

Mepivacaine from In vitro diffusion media

Selection of suitable drug diffusion media and development of an analytical method for

quantification of mepivacaine from the diffusion media: Suitable diffusion media is the

pre requisite for the in vitro diffusion studies. Since the formulations are intended for

topical applications, the receptor media was selected such that its pH simulates to the skin

pH. Phosphate buffer solution of pH 6.8 was used as the receptor media for in vitro

diffusion studies.

The objective of validation was to demonstrate the suitability of developed method for

quantification of drug from the in vitro diffusion media i.e., the method should be

sensitive enough to detect low concentrations and should be reproducible and linear. The

method was validated for the following parameters.

A. Linearity

The linearity of the method was investigated by studying the linear regression of the areas

under the peaks obtained by analyzing solutions with mepivacaine concentrations in the

range of 1.0 – 10.0 µg/mL.

B. Precision

Intra-day precision: The intraday precision was evaluated (n = 3) at three

concentration levels (1.0, 5.0 and 10.0 µg/mL) of mepivacaine and % relative standard

deviation of three values was calculated.

Fig 2.21: Standard Calibration curve of

MH in PBS pH 6.8

Fig 2.22: Standard Calibration curve of

MB in PBS pH 6.8


79

Inter-day precision: Concentrations of 1.0, 5.0 and 10.0 µg/mL of mepivacaine were

analyzed on three different days for assessing inter day precision. Three triplicates per

concentration were injected into the HPLC.

Table 2.27:Statistical data showing intra-day precision of method for quantitation of

mepivacaine during in vitro diffusion study.

Concentration

µg/mL

Average

area

Predicted

average

% RSD SE R2

Day 1

0.1 102.59 102.65 0.1456 0.1412 0.9998

0.6 321.89 321.90 0.0765 0.2341

1.0 609.98 610.21 0.0165 0.2781

Day 2

0.1 103.27 103.30 0.2134 0.2121 0.9999

0.6 322.45 322.50 0.0856 0.3843

1.0 610.32 610.45 0.0201 0.3118

Day 3

0.1 102.98 103.45 0.2351 0.3138 0.9999

0.6 322.88 323.05 0.0961 0.3112

1.0 610.76 610.95 0.0271 0.2890

Table 2.28: Statistical data showing inter-day precision of method for quantitation of

mepivacaine during in vitro diffusion study

Concentration

µg/mL

Average

area

Predicted

average % RSD R

2

0.1 102.59 103.05 0.1027

0.9999 0.6 321.89 322.15 0.0573

1.0 609.98 610.12 0.0654

2.5.5.3 Method Development and Validation for Quantification of Mepivacaine from

Skin Homogenate.

Measurement of drug concentration in the skin is essential during the development of

topical products. Active accumulation in the skin with minimal or no systemic absorption

is desired. This requires the development of a robust and sensitive method to extract and

quantify the drug in the skin in order to predict skin targeting. An analytical method was

developed and validated for quantification of mepivacaine in porcine and rat skin samples

for ex vivo and in vivo penetration studies. Porcine ear skin was used for ex vivo skin

penetration studies. Therefore initial method development was carried out with porcine


80

ear skin. Further the method was extended to rat skin homogenate and standard curves

were prepared in homogenates of rat skin layers (epidermis and dermis).

The aim of this part of research work was to develop a simple method to extract and

quantify mepivacaine from porcine and rat skin samples in order to predict if adequate

amounts of active have reached viable layers of skin and to assay the drug using HPLC

method of analysis.

Selection of Internal Standard: Internal standards are commonly used in quantitative

bioanalysis particularly in HPLC / MS based bio-analysis. By finding a good internal

standard and using analyte / IS response ratios for quantitation, variations in absolute

responses other than those related to analyte concentration could be corrected, which help

maintain the accuracy of quantitative results. Various chemical compounds were

investigated for their suitability as internal standard along with mepivacaine.

Method development for extraction of mepivacaine from skin homogenate: The

method was developed in model of porcine ear skin, as porcine ear skin is well accepted

as a decent facsimile of the human counterpart because of its physiological and structural

similarity to human skin [Schmook FP; 2001; Herkenne C; 2008, Teichmann A ; 2006].

A) Skin preparations: Porcine ear skin

The porcine ears were obtained post sacrifice from the local abattoir (VileParle,

Mumbai), before the pig carcass was exposed to the normal high temperature cleaning

procedure. The ears were stored at -20oC and thawed slowly at 4

oC before gently cutting

the hair and removing full thickness skin from the back of the ears using a scalpel without

damaging or scratching the surface in order to ensure the integrity of the skin barrier.

Subcutaneous tissue was carefully removed with a scalpel, and the skin was cut into

appropriate pieces and was stored at -20oC until further use (Not more than 2 weeks).

B) Extraction of drug from skin homogenate

Mepivacaine was extracted from the skin by cutting it into very fine pieces using scissors

and homogenized with tissue homogenizer separately in methanol (5ml) as mepivacaine

has very good solubility in methanol. The homogenate was then vortex mixed and further

homogenized by ultra sonication for complete extraction of actives. The resulting

homogenate was filtered using 0.2µ membrane filter to get clear solution and active

content in the filtrate was determined after appropriate dilution.


81

In order to confirm the reliability, accuracy and precision of the method, several

parameters were investigated according to ICH guidelines for analyzing mepivacaine in

porcine ear skin samples.

A. Specificity

The specificity of the method was verified by evaluating the interference of skin

components with the assay of active, mepivacaine. To determine the method specificity,

the control skin samples were treated by the same procedure as that to extract actives and

the spectra obtained from these injections were compared to the skin homogenate spiked

with mepivacaine.

B. Linearity

Linearity curves to estimate mepivacaine in skin samples were constructed by assaying

skin homogenates spiked with mepivacaine in the concentration range of 2.0 to 20 µg/ml.

These standard solutions were vortexed for 30 secs and centrifuged at 3000 rpm for

10min. The supernatants were collected and then evaporated. The residue collected after

evaporation was reconstituted to 10 mL with mobile phase. The diluted samples were

filtered; injected into the HPLC and chromatograms were recorded at 210 nm. An

appropriate concentration of internal standard was selected for spiking the homogenates

based on preliminary studies. The final solutions were injected in triplicate. The

calibration curve was obtained by plotting peak area ratio of mepicavaine v/s

concentration. The linearity was repeated on three different days. The regression

coefficient was calculated for each day.

C. Precision

Intra-day precision

The intra-day precision was determined by sequential analysis at three concentration

levels of mepivacaine (2.0, 10.0 & 20 µg/mL). Each sample was analyzed at three

different times a day, totaling to nine analyses in a day.

Inter-day precision

Three different concentrations 2.0, 10.0 & 20 µg/mL of mepivacaine were analyzed on

three different days. Three replicates per concentration were injected in to the HPLC. The

SD and standard error of mean (SE) were compared as a measure of scatter of method

precision.


82

Repeatability

System repeatability was determined by injecting 10 replicates of 4.0 µg/mL and 6.0

µg/mL of mepivacaine solution respectively which were prepared by spiking the

appropriate concentrations of drug and internal standard into the skin homogenate

followed by extraction. The entire procedure was repeated on three different days and

the relative standard deviation (% RSD) was calculated to determine the deviation of

each measurement from mean value for each day.

Recovery

High recovery of the drug from the homogenate matrix is a desirable outcome of sample

preparation and is therefore an important characteristic of the extraction procedure. The

absolute recovery was determined as the ratio of response measured for the spiked

sample (in homogenate matrix) treated according to the extraction procedure to that of a

non biological sample spiked with the same quantity of the drug and directly injected into

the chromatographic system. Three different concentration levels (2.0, 10.0 and 20.0

µg/mL) were investigated for determination of extraction recovery.

Fig 2.25: Calibration Curve of MB in the presence of porcine skin matrix

Table 2.29: Statistical data showing intra-day precision for extraction of mepivacaine

from porcine ear skin

Fig 2.23: Chromatogram showing analysis

of blank porcine ear skin homogenate.

Fig 2.24: Chromatogram showing

analysis of drug loaded porcine ear

skin homogenate.


83

Concentration

µg/mL

Average

area

Predicted

average

% RSD R2

Day 1

2.0 0.3612 0.3620 0.3882 0.9998

8.0 1.3789 1.3795 0.2291

10.0 2.5871 2.5882 0.2339

Day 2

2.0 0.3725 0.3730 0.3551 0.9996

8.0 1.3890 1.3895 0.2392

10.0 2.5915 2.5928 0.2531

Day 3

2.0 0.3911 0.3925 0.3882 0.9997

8.0 1.3995 1.4004 0.2748

10.0 2.5976 2.5985 0.2411

Table 2.30: Statistical data showing inter-day precision for extraction of mepivacaine

from porcine ear skin

Concentration

µg/mL

Average

area

Predicted

average

SD % RSD R2

2.0 0.3512 0.3555 0.0035 0.9862 0.9987

8.0 1.3201 1.3236 0.0193 1.0931

10.0 2.5780 2.5809 0.0249 0.8947

Table 2.31: Statistical data showing repeatability of mepivacaine from porcine ear skin

Average Area Predicted Area SD % RSD

Day 1 0.6592 0.6389 0.0045 0.7375

Day 2 0.6639 0.6385 0.0049 0.7278

Day 3 0.6712 0.6321 0.0048 0.6911

Table 2.32: Recovery data of mepivacaine in spiked porcine skin samples

Conc

µg/mL

Non biological Spiked

sample (µg/mL)

Tissue homogenate

matrix

%

Recovery

2.0 0.4331 0.4550 96.34

8.0 1.3254 1.3431 98.45

10.0 2.6125 2.6250 97.59


84

Table 2.33: Validation parameters of the developed HPLC method using porcine skin


LOD (ng/ml) 20

LOQ (ng/ml) 60

Linearity

Range (ng/ml) 50-500

Regression values 0.9992

Demonstration of suitability of extraction method for quantification of mepivacaine

from rat skin:

A. Skin preparation: Rat skin was depilated to remove hair. The skin was excised and

carefully dissected making sure that the subcutaneous fat was maximally removed and

stored at -40oC until further use.

B. Extraction of drug from skin homogenate: The active mepivacaine from the rat skin

layer (epidermis and dermis) homogenate was extracted using the same method as

described in the section of porcine ear skin.

C. Validation of the developed method

Validation of method for extraction of mepivacaine from rat skin layers (epidermis and

dermis) was carried out as described under previous section. The amount of active in

stratum corneum was analyzed by tape stripping.

Fig 2.28: Calibration Curve of MB in the presence of rat skin matrix.


analysis of blank rat skin homogenate


of drug in rat skin homogenate


85

Table 2.34: Validation parameters of the developed HPLC method using rat skin matrix


LOD (ng/ml) 20

LOQ (μg/ml) 60

Linearity

Range (ng/ml) 50-500


2.5.5.4 Method Development and Validation for drug extracted from Tape Strips

used for in vivo Penetration Studies

Sequential tape-stripping of the stratum corneum (SC) of skin allowed horizontal

fractions of membrane to be obtained. The method involves extracting the tape strips in

suitable solvent so as to recover and quantify the absorbed active. The method of

quantification depends on the nature of the active and the amount present on the tape

strips. The key criteria’s are that the extraction process should be efficient and

reproducible, and that it should be free from interference from components of the SC

and/or the tape adhesive. Therefore the aim was to develop and validate a standardized

method for assessment of active compound from the skin by tape stripping method and

HPLC analysis procedure which should be sensitive enough for determination of active in

extraction samples from adhesive tapes.

Development of extraction procedure of mepivacaine from tape strips:

Selection of adhesive Tapes: Selection of adhesive tape with appropriate properties is a

necessary prerequisite for accurate studies based on stratum stripping for determination

of an active in the stratum corneum. The prerequisite of the tape used in the present

experimental part was to show no absorbance at wavelength used for quantification of

mepivacaine thus preventing interferences.

Extraction Procedure: To determine HPLC specificity, the tapes were extracted with

5ml extraction medium in a stoppered 25 ml conical flask. The contents were then

sonicated and filtered. The filtered aliquot solution was injected in to the HPLC column

without further dilution. The chromatograms obtained from these injections were

compared with those obtained for the standard drug solution.


86

Development of extraction procedure from 3M Micropore tape strips:

Sr No Extraction procedure from 3M Micropore tape strips

1. Tape strips were immersed in chloroform, sonicated and the supernatant

was filtered through 0.2µ membrane filter and injected into HPLC

2. Tape strips were immersed in methanol, sonicated and the supernatant was

filtered through 0.2µ membrane filter and injected into HPLC

3. Tape strips were immersed in IPA, sonicated and the supernatant was

filtered through 0.2µ membrane filter and injected into HPLC

Sample Preparation: The extraction procedure was validated by spiking tape stripped

samples of untreated SC with known amounts of active chosen to represent the expected

range of concentrations encountered during the in vivo experiments.

A. Specificity

To assure that there is no interference of matrix constituents of the tape (e.g. adhesive,

polymeric film on the tape) and skin components (such as lipids, proteins) with the active

drug peak, tapes stripped from untreated rat skin were compared with the tape stripped

from the untreated skin spiked with standard mepivacaine solution.

B. Precision

Inter day variation: The precision under the same operating conditions over a short

interval of time was determined by sequential analysis of spiked solutions at three

concentration levels (0.5, 1.5 and 2.5 µg/ml). Each sample was analyzed at three different

times of a day, totaling to nine analyses a day.

Inter day variation: Three replicates of three concentration levels 0.5, 1.5 and 2.5 µg/ml

were analyzed on three separate days for the estimation of intermediate precision and the

measure of scatter of method was computed.

System repeatability: Tapes were spiked with mepivacaine solution to yield loading dose

of 1.0 µg/ml on the tapes. The samples were injected six times and the chromatograms

were recorded. The experiment was repeated on three different days and the relative

standard deviation was calculated for each day. The repeatability data is shown in Table

2.37.


87

C. Linearity

Linearity of the analytical method was determined by spiking the tape strips with stock

solutions of mepivacaine doses in the range of 0.5 to 5.0 µg/mL.

D. Recovery

The demonstration of the method’s extraction efficiency was achieved using the response

measured for the spiked sample (skin stripped tapes) treated according to the whole

analytical procedure to that of a non biological sample spiked with the same quantity of

the drug and directly injected into the chromatographic system. Three different

concentration levels (0.5, 1.5 and 2.5 µg/mL) were investigated for determination of

extraction recovery. Results are as in Table 2.38

Fig 2.31: Calibration Curve of MB in the presence of tape strip sample

Fig 2.29: Chromatogram showing peaks

of blank tape strip sample


peaks of MB+IS in presence of tape

strip sample


88

Table 2.35: Statistical data showing intra-day precision of method for extraction of

mepivacaine from tape strip

Concentration

µg/mL

Average area Predicted

average

% RSD R2

Day 1

0.5 625.21 625.56 0.4362 0.9998

1.5 1477.12 1478.67 0.3982

2.0 2832.67 2840.50 0.3313

Day 2

0.5 627.45 627.45 0.4578 0.9997

1.5 1480.49 1480.49 0.3321

2.0 2838.46 2838.46 0.2291

Day 3

0.5 631.87 632.45 0.4118 0.9999

1.5 1488.10 1489.22 0.3391

2.0 2845.39 2848.21 0.2274

Table 2.36: Statistical data showing inter-day precision of method for extraction of

mepivacaine from tape strips

Concentration

µg/mL Average area Predicted average % RSD R

2

0.5 620.21 621.50 0.5690

0.9997 1.5 1480.39 1485.12 0.6112

2.0 2898.54 2899.79 0.5331

Table 2.37: Statistical data showing repeatability of method for extraction of mepivacaine

from tape strips

Conc µg/mL Average Area Predicted Area % RSD

Day 1 1.0 1175.36 1175.89 0.7762

Day 2 1.0 1178.43 1178.50 0.8112

Day 3 1.0 1180.12 1180.26 0.7743

Table 2.38: Recovery data for estimation of mepivacaine in spiked tape strip samples

Conc

µg/mL

Non biological Spiked

sample (µg/mL)

Tissue homogenate

matrix

%

Recovery

Average %

recovery

0.5 621.45 621.65 98.34

97.42 1.5 1483.48 1483.60 97.47

2.0 2894.34 2895.23 96.45


89

2.5.5.5 Method Development and Validation of Mepivacaine extracted from Plasma

Measurement of drug concentration in skin is essential in the development of topical

formulations. Active accumulation in the skin with minimal or no systemic absorption is

desired. The aim of this part of the research work was to develop a sensitive and reliable

bioanalytical HPLC method for the estimation of mepivacaine in rat plasma during

pharmacokinetic studies. Thus the purpose of this work was to develop and validate the

method for extraction of the actives from plasma to prove that the pharmacological effect

produced was due to local action of the actives and no significant systemic absorption

was observed.

Preparation of Standard Solutions: The stock (100 µg/mL) and sub-stock solutions

containing mepivacaine with concentrations of 1, 5, 10, 15 and 20 µg/mL each were

prepared using methanol as solvent. Internal standard stock solution was also prepared at

a concentration of 5 µg/mL using the same solvent.

Development of Extraction Procedure for Extraction of mepivacaine from Plasma:

The purpose was to develop suitable extraction procedure for the active and the Internal

standard (IS) from plasma. Mepivacaine was separated from the plasma matrix using

liquid–liquid extraction with an organic solvent under alkaline conditions. Plasma (0.5

ml) spiked with pre determined quantity of drug solution (or IS) was mixed with

sufficient quantity of alkaline (NaOH) solution, and organic solvent (DCM). The samples

were vortexed for 20 secs, followed by centrifugation at 2000 rpm for 3 minutes. The

upper aqueous layer was aspirated to waste. The organic layer was transferred into a

conical centrifuge tube and evaporated to remove the traces of organic phase till

completely dry. The samples were reconstituted in acetonitrile to dissolve the residue.

The final concentration of drug was 100, 150, 200, 250, 300, 350, 500 ng/mL and 100

µg/mL of IS in these solutions. The solution was filtered, injected into HPLC and the

chromatograms were recorded at 210 nm.

A. Linearity

It was determined by spiking the plasma sample with stock solution of mepivacaine in the

range of 0.05 to 0.5 µg/ml and with appropriate concentration of Internal standard. The

drug was extracted and analyzed as described earlier. The calibration curve was obtained

from the least square linear regression. The regression line was used to calculate the


90

respective concentrations of drug in the plasma obtained from the rats 24 h after

application of topical dosage forms on the rats skin surface.

B. Recovery

The demonstration of the method’s extraction efficiency was achieved using the response

measured for the biological sample spiked treated according to the whole analytical

procedure to that of a non biological sample spiked with the same quantity of the active

and injected into the chromatographic system. For determination of extraction recovery

three different concentration levels, (0.08, 0.12 and 0.22 µg/ml) were studied.

C. System precision

It was determined by performing six repeated analysis of working standard solution of a

concentration of 100ng/mL. Intermediate precision was assessed by analyzing three

replicates of reference solutions at three levels (0.08, 0.12 and 0.22 µg/mL) on the same

day and on three consecutive days.

D. Accuracy

The accuracy of the analytical method was determined by replicate analysis of three

quality control samples containing known amount of analyte (0.08, 0.12 and 0.22

µg/mL).

Fig 2.34: Calibration Curve of MB in the presence of rat plasma as biological matrix.


of drug in presence of plasma.

Fig 2.32: Chromatogram showing peaks

of blank plasma sample


91

Table 2.39: Validation parameters of the developed HPLC method using rat plasma as

biological matrix


LOD (ng/ml) 20

LOQ (ng/ml) 60

Linearity

Range(ng/ml) 50-500


System precision

Theoretical peak area ratio 2.93

Observed peak area ratio 2.89

% RSD 0.98

Table 2.40: Precision and accuracy results for developed HPLC method for mepivacaine

using rat plasma as biological matrix

Sample

n=3

Theoretical

conc

(ng/mL)

Intra-day

precision

(% RSD)

Inter-day

precision

(% RSD)

Percent

Accuracy

Mean %

Accuracy

+ % RSD

%

Recovery

Mean %

Recovery

+ %RSD

Low

Q.C 80 0.63 1.51 99.02

99.67 ±

0.70

99.45

99.45 ±

0.60

Medium

Q.C 120 0.27 1.38 100.63 98.86

High

Q.C 220 0.47 0.45 99.36 100.03

*Q.C - Quality Control


92

2.5.6. Results and Discussion:

2.5.6.1. UV–visible spectrophotometric method for estimation of Mepivacaine HCl,

Mepivacaine base and Lidocaine HCl for drug assay in semisolid dosage forms

In the present work, U.V spectrophotometric method for the quantitation of Mepivacaine

HCl and base and Lidocaine in topical dosage form was developed for routine analysis.

The method was developed by using phosphate buffer solution pH 6.8 and methanol as

solvents as the drug showed good solubility in both the solvent systems. In proposed

method, absorption maxima was obtained at 263 nm for mepivacaine base & HCl and

264 nm for lidocaine and the calibration curve obeyed Beer-Lambertz law in the

concentration range of 100-450 μg/ml with correlation coefficient (r²) of 0.9999 and

0.9998 in phosphate buffer pH 6.8 and methanol respectively. The developed method was

validated according to ICH guidelines for validation of analytical procedures. Limit of

detection was found to be 20 μg/ml and limit of quantification was 60 μg/ml for

mepivacaine HCl and base. The low values of percentage relative standard deviation

showed that the developed method was precise. All statistical data proved validity of

proposed method, which can be applied for assay of mepivacaine. Although the proposed

method was found to be linear, precise and accurate, it is not very sensitive for the

quantification of mepivacaine from in vitro/ex vivo diffusion media. Hence a more

sensitive HPLC method was developed for the analysis of mepivacaine HCl and base

from topical formulations.

2.5.6.2. Analytical Method Development and Validation for Quantification of

Mepivacaine from In vitro diffusion media

The initial mobile phases tried were based on published data on mepivacaine as shown in

Table 2.25. However a well resolved peak of drug could not be obtained. The developed

mobile phase used for the quantitation of mepivacaine from the diffusion media consisted

on acetonitrile: phosphate buffer solution pH 6.8 (6:4 v/v). The validation was carried out

to demonstrate the suitability of the developed method for quantitation of mepivacaine

from the in vitro diffusion media i.e., the method should be sensitive enough to detect

low concentrations of the active and should be repeatable and linear. The analytical

method was validated for linearity and precision.


93

Linearity: The linear regression coefficients for the constructed calibration curves of

mepivacaine demonstrated linearity with r2

value greater than 0.999.

Precision: Intra-day and Inter-day precision of mepivacaine analyzed at three different

concentrations showed % RSD values < 2 as shown in Table 2.27 and 2.28. This

indicated that the developed method for quantification of drug from the in vitro diffusion

media was precise.

Thus an analytical method for in vitro diffusion studies was developed and found to be

selective, sensitive, linear and precise.

2.5.6.3. Analytical Method for extraction of drug from Porcine Ear Skin.

Mepivacaine could be easily extracted from the porcine ear skin using tissue

homogenizer for homogenizing the skin sample and methanol as solvent for extraction

since the drug has excellent solubility in methanol. The suitability of the method was

further verified by performing the detailed validation of the method as per ICH

guidelines.

Specificity: The control skin sample HPLC spectra were compared with HPLC spectra

from mepivacaine spiked skin sample. The retention time of the drug was recorded at 5.8

min. In the chromatogram of the skin extracts, skin components did not interfere with the

peak of interest (Fig 2.23 and 2.24). Since no interference between the drug and skin

matrix components was observed in the HPLC spectra, the method was proved to be

selective and specific.

Linearity: Calibration curve constructed for mepivacaine by plotting the graph of

concentration versus mepivacaine area was found to be linear in the range of 2.0 to

10µg/ml as shown in the Fig 2.25. The analytical method showed a regression coefficient

greater than 0.999 on all the three days. Thus the linear regression analysis demonstrated

acceptability of the method for quantitative analysis of mepivacaine in the skin samples.

Intra-day and Inter-day Precision: The observed lower values of relative standard

deviation, lower % RSD values <2, at both, intra-day and inter-day analysis indicated the


94

method to be precise. It showed the acceptability of the method with adequate intra-day

and inter-day precision (Table 2.29 & 2.30).

Repeatability: SD, % RSD and SE displayed low variance for three separate days for

mepivacaine as shown in the Table 2.31. This demonstrates the method to be repeatable

for the analysis of mepivacaine from the skin homogenate.

Recovery: The recovery was calculated from the mepivacaine concentration with the non

skin sample and compared with the spiked skin homogenate. The mean recovery of

mepivacaine was 96.34%, 98.45% and 97.59% at concentrations of 2.0, 8.0 and 10.0

µg/mL respectively (Table 2.32). The average recovery over the entire analytical range

was 97.46%. From the recovery rates, it can be concluded that the extraction procedure

provided a reliable quantitative determination of the drug in skin extracts.

2.5.6.4. Analytical Method for extraction of drug from Rat Skin.

The linear regression coefficients and calibration curves in the range of 0.1 to 0.5 µg/mL

of mepivacaine showed that the extraction method from epidermis and dermis of rat skin

homogenate is linear with R2

value greater than 0.998 on three separate days. The

chromatogram showing analysis of blank and drug in rat skin homogenate are shown in

Figs 2.26 and 2.27 and the standard calibration curve of MB in the presence of rat skin

matrix is shown in Fig 2.28.

2.5.6.5. Analytical Method for determination of drug content from Tape Strips.

The 3M Micropore tapes were investigated with the aim to extract the drug efficiently

and selectively and to eliminate the interference of tape constituents such as adhesives

and polymer with the peak of interest. When Chloroform and isopropyl alcohol was used

as extraction media, interference with the active peak was observed. However, when

methanol was used as extraction media, no interference due to tape strip constituents and

skin components was observed at the Rt of the active, mepivacaine.

Specificity: The method developed here proved to be selective since the retention times

for other compounds present in the skin or in the adhesive tape, analyzed under the same

chromatographic conditions, was not similar to those obtained for mepivacaine showing


95

no interference between them and the tape matrix constituents or the skin components,

thereby validating the specificity of the technique as shown in the Figs 2.29 and 2.30.

Intra-day and Inter-day Precision: The observed lower values of relative standard

deviation, lower % RSD values <2, at both, intra-day and inter-day analysis indicated the

method to be precise. It showed the acceptability of the method with adequate intra-day

and inter-day precision (Tables 2.35 & 2.36).

Repeatability: Table 2.37 shows lower % RSD values for mepivacaine thus confirming

that the method could be repeatable and reproducible and the precision was found to be

acceptable for analytical purposes.

Linearity: Chromatograms recorded over a concentration range of 0.5 to 5.0 µg/mL

yielded a linear relationship between the peak area and the concentrations. The method

demonstrated the acceptable linearity with regression coefficient greater than 0.996. The

typical calibration curves obtained for mepivacaine are shown in Fig 2.31.

Recovery: The mean recovery for mepivacaine from the tape strips was found to be

98.34% at a concentration of 0.5 µg/mL, 97.47% at a concentration of 1.5 µg/mL and

96.45% at a concentration of 2.0 µg/mL.

Therefore, the average % recovery over the entire analytical range was 97.42. The

recovery data is summarized in Table 2.38. High recovery of the drug from the tissue

matrix is a desirable outcome of sample preparation and is therefore important

characteristic of extraction procedure. From the recovery rates, it could be concluded that

the extraction procedure provides a reliable quantitative determination of drug in tape

extracts.

2.5.6.6. Analytical Method for extraction of drug from Plasma.

Mepivacaine was extracted from the plasma matrix using liquid–liquid extraction using

dichloromethane as an organic solvent under alkaline conditions.

Linearity: Chromatograms recorded over a concentration range of 0.05 to 0.5 µg/mL

yielded a linear relationship between the peak area and concentration. The method


96

demonstrated acceptable linearity with regression coefficient greater than 0.998 as shown

in the Fig 2.34.

Recovery: The recovery study of mepivacaine from the plasma samples was studied at

three concentrations of 0.08 µg/mL, 0.12 µg/mL and 0.22 µg/mL and the mean recovery

was found to be 99.45%, 98.86% and 100.03% respectively. From the recovery data of

mepivacaine as depicted in Table 2.40, it could be concluded that this extraction

procedure was efficient and gave good recoveries.

The LOD and LOQ of mepivacaine in rat plasma matrix were found to be 20 and 60

ng/mL with mean % accuracy of 99.67%. Hence, the analytical method developed and

validated for the quantitation of mepivacaine in rat plasma matrix was sensitive, precise

and accurate.

This developed and validated HPLC method will be useful for estimation of drug from

topical formulations.


97

2.6. Forced Degradation Studies on Mepivacaine

The potential impurities of Mepivacaine HCl reported are 2, 6-Picoloxylidide

(intermediate) and -2,6-Pipecoloxylidide (side product). The Degradation impurities of

mepivacaine reported are Xylidine (Hydrolysis product), -1-Methyl-2-

piperidinecarboxylic acid (Hydrolysis product).

There is an increased interest in the identification and control of potentially genotoxic

impurities. In 2007, the European Medicines Agency (EMEA) issued guidelines on the

limits of genotoxic impurities [EU EMEA/CHMP Guidelines], and the Food and Drug

Administration (FDA) issued draft guidance on the same subject in December 2008 [US

FDA/CDER, Guidance]. A validated analytical procedure was used which was capable of

determining mepivacaine in the presence of formulation excipients and potential

degradation products was used.

2.6.1. Standard preparation

About 10 mg of mepivacaine reference standard was accurately weighed and dissolved in

the mobile phase to make 100 ml solution. A 10-ml aliquot of the resulting solution was

further diluted to 100 ml with mobile phase.

2.6.2. Test preparation

About 500-mg, accurately weighed, portion of the mepivacaine gel was dispersed by

sonication for 10 min in 50 ml of mobile phase and diluted to 100 ml with the mobile

phase. The solution was filtered using a 0.45-µm filter membrane discarding the first 15

ml. Samples of the mepivacaine gel and placebo gel were stressed under the following

conditions, and test preparations were prepared using the stressed samples.

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98

2.6.3. Thermal degradation

Mepivacaine gel was weighed and placed in a sealed glass tube. The glass tube was

placed into a heated tube furnace (Lindberg/Blue) at 150◦C for 4 hrs. The glass tube was

then allowed to cool to room temperature. Then the sample was analyzed by HPLC.

2.6.4. Photo-degradation

Mepivacaine gel samples were spread as thin layers on the inside walls of two quartz UV

cells. The cells were exposed uncovered, sample side up, to UV (254 and 365 nm)

(output: 300mW/cm2 at 6”) and white light under ambient conditions for 48 hrs. After 48

hrs, the light source was removed and sample was analyzed.

2.6.5. Oxidative degradation

Hydrogen peroxide (3%, 3mL) was added to diluted mepivacaine gel sample. This

sample was then analyzed after 4 h to estimate degradation of mepivacaine.

2.6.6. Acid degradation

Hydrochloric acid (5N, 3mL) was added to gel sample and heated at 60◦C for 4 h. After 4

h, the solution was brought to ambient temperature and to this solution, 6mL of

acetonitrile was added and drug was extracted.

2.6.7. Base degradation

NaOH (5N, 3mL) was added to gel sample and heated at 60◦C for 4 h. After 4 h this

solution was brought to ambient temperature and to this solution, 6mL of acetonitrile was

added and drug was extracted. Results of the test are as shown in Figs 2.35-2.39.

Fig 2.36: Chromatogram of

Mepivacaine exposed to U.V. Light for

48hrs.

Fig 2.35: Chromatogram of Reference

Mepivacaine


99

Results and Discussion

The stability and degradation profiles of mepivacaine in gel samples were evaluated with

the long-term objective of developing stable topical formulations of this drug.

Chromatogram of mepivacaine extracted in the gel sample at 210 nm showed no

interferences between the drug and excipient peaks. The stability profile of mepivacaine

gel samples at high temperature and light conditions was monitored by high performance

liquid chromatography (HPLC). Forced degradation of mepivacaine gel sample was

performed at extreme heat, light, acidic (pH<2.0) and alkaline (pH>10.0) conditions and

by addition of hydrogen peroxide (oxidizing agent). Mepivacaine was very stable to acid

and base treatment, no degradation product was seen. Also, it was found stable to

oxidation. There were no significant changes in the chromatograms obtained with

mepivacaine gels subjected to oxidative, heat, light, acid or base stress, when compared

to the chromatograms obtained with the reference sample. The physical appearance of the

gel, however, changed when it was subjected to heat stress—the gel appeared to lose its

viscosity.

The developed HPLC procedure separated the excipients and other peaks from the

mepivacaine peak and will be utilized for the analysis of mepivacaine in compounded

mepivacaine topical formulations.

Fig 2.39. Chromatogram of

Mepivacaine after acid treatment.

Fig. 2.40. Chromatogram of

Mepivacaine after alkaline treatment.

Fig 2.37: Chromatogram on thermal

degradation.

Fig 2.38: Chromatogram of

Mepivacaine on oxidation

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